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  • MicroRNAs in the moss Physcomitrella patens

    Tzahi Arazi

    Received: 13 December 2010 / Accepted: 25 February 2011

    Springer Science+Business Media B.V. 2011

    Abstract Having diverged from the lineage that lead to

    flowering plants shortly after plants have established on

    land, mosses, which share fundamental processes with

    flowering plants but underwent little morphological chan-

    ges by comparison with the fossil records, can be consid-

    ered as an evolutionary informative place. Hence, they are

    especially useful for the study of developmental evolution

    and adaption to life on land. The transition to land exposed

    early plants to harsh physical conditions that resulted in

    key physiological and developmental changes. MicroRNAs

    (miRNAs) are an important class of small RNAs (sRNAs)

    that act as master regulators of development and stress in

    flowering plants. In recent years several groups have been

    engaged in the cloning of sRNAs from the model moss

    Physcomitrella patens. These studies have revealed a

    wealth of miRNAs, including novel and conserved ones,

    creating a unique opportunity to broaden our understanding

    of miRNA functions in land plants and their contribution to

    the latters evolution. Here we review the current knowl-

    edge of moss miRNAs and suggest approaches for their

    functional analysis in P. patens.

    Keywords AGO1 DCL1 Development Evolution Gametophyte MicroRNA Moss Physcomitrella patens Stress


    MicroRNAs, are genome-encoded noncoding RNAs of*21nucleotides (nt) in length that act as repressors of target genes

    in animals and plants (Bartel 2004). A mature miRNA and its

    passenger strand (miRNA*) are derived from opposing arms

    of a hairpin precursor (pre-miRNA) found in a longer pri-

    mary transcript (pri-miRNA) that is transcribed from MIR

    genes by RNA polymerase II (Chen 2008). In Arabidopsis

    thaliana, release of a pre-miRNA from the pri-miRNA and

    its subsequent processing into a mature miRNA/miRNA*

    duplex occurs via at least two cleavage steps, which are

    catalyzed by the RNaseIII-type enzyme DICER-LIKE 1

    (DCL1) (Kurihara et al. 2006; Kurihara and Watanabe 2004;

    Park et al. 2002) assisted by HYPONASTIC LEAVES1 and

    SERRATE (Dong et al. 2008; Kurihara et al. 2006). Fol-

    lowing its release, the miRNA/miRNA* duplex is methyl-

    ated by HUA ENHANCER 1 (Yu et al. 2005). Then, its two

    strands are separated and the single-stranded mature miRNA

    is specifically sorted into an ARGONAUTE (AGO) protein

    that form the core of an effector complex called RNA-

    induced silencing complex (RISC) (Mi et al. 2008). AGO1 is

    considered the major miRNA-RISC. It performs slicer

    activity that cleaves miRNA targets (Baumberger and

    Baulcombe 2005; Qi et al. 2005) and preferentially associ-

    ates with 21-nt-long sRNAs that have a 50 terminal uridine(Mi et al. 2008; Montgomery et al. 2008), features which are

    characteristic of most plant miRNAs. Loaded miRNAs guide

    RISC to nearly complementary target sequences resulting in

    their cleavage (Llave et al. 2002; Tang et al. 2003) or

    translation repression (Brodersen et al. 2008; Chen 2004).

    Accumulating evidence on miRNA functions in A. thaliana

    and other model flowering plants suggests that they

    play critical roles in vegetative and reproductive develop-

    ment (Chen 2009), nutrient homeostasis, response to

    T. Arazi (&)Institute of Plant Sciences, Agricultural Research Organization,

    Volcani Center, PO Box 6, Bet Dagan 50250, Israel

    e-mail: tarazi@agri.gov.il


    Plant Mol Biol

    DOI 10.1007/s11103-011-9761-5

  • environmental stresses (Sunkar 2010), and autoregulation of

    the miRNA pathway itself (Vaucheret et al. 2006; Xie et al.


    Bryophytes are believed to have shared a common

    ancestor with flowering plants *400 million years ago(MYA) (Kenrick and Crane 1997). Physcomitrella patens

    (Bryopsida) is a monoecious moss that has emerged as a

    useful model plant mainly because it is easily transformed

    and performs efficient homologous recombination, which

    allows the study of gene function by targeting gene dis-

    ruptions or replacements (Cove 2005; Schaefer 2001).

    Moreover, its complete genome sequence (*511 Mb) wasrecently published (Rensing et al. 2008) and powerful

    molecular tools are available (Frank et al. 2005a). Unlike

    vascular plants (ferns and seed plants), the life cycle of

    P. patens like all mosses is dominated by a haploid

    gametophyte phase (reviewed in Reski 1998) making the

    analysis of engineered loss-of-function P. patens mutants

    straightforward. Furthermore, the P. patens gametophyte

    has simple tissue morphology with only a few cell types,

    which facilitate the characterization of abnormal mutant

    phenotypes and the study of plant development (Prigge and

    Bezanilla 2010).

    The development of the dominant P. patens gameto-

    phyte, which is larger and more complex than the sporo-

    phyte, can be divided into two distinct stages: the

    protonema (juvenile gametophyte) and the gametophore

    (adult gametophyte) (Fig. 1). The protonema, generated by

    spore germination, is composed of filaments of cells that

    extend by successive divisions of their tip cell (Menand

    et al. 2007). Young protonema filaments have assimilatory

    functions and consist of chloronema cells that are densely

    packed with large chloroplasts and have perpendicular cell

    walls (Fig. 1a). These filaments extend until, in response to

    increases in light (Cove and Ashton 1988) and auxin (Johri

    and Desai 1973) their tip cells differentiate into caulonema

    cells, which are longer, divide more often, contain fewer

    smaller chloroplasts and have oblique cell walls (Fig. 1b).

    Soon after the division of a caulonema tip cell, an initial

    cell is formed in the second subapical cell. In the presence

    of cytokinin this initial cell, instead of producing a lateral

    filament, will divide and produce a bud, marking the

    transition from juvenile to adult gametophyte stage

    (Schumaker and Dietrich 1998) (Fig. 1c). By meristematic

    growth, this bud forms a leafy gametophore on which

    female and male gametangia will later develop (Fig. 1d).

    Once fertilized, the zygote will develop into a tiny diploid

    sporophyte that produces haploid spores (Fig. 1e).

    Cloning efforts have identified numerous miRNAs from

    various P. patens gametophyte tissues, many of which

    target regulatory genes. Furthermore, certain miRNA

    families and their corresponding targets have been found to

    be conserved with flowering plants, suggesting a common

    origin of miRNA-regulated pathways in land plants (Axtell

    and Bowman 2008). In this article, we review what is

    known about P. patens miRNAs and discuss how miRNA

    functions and regulatory roles may be elucidated in this

    unique model plant.

    miRNA discovery in P. patens

    The first experimental evidence of miRNA-guided target

    cleavage in a non-flowering plant was provided by Floyd

    Fig. 1 A cartoon of major stages in P. patens gametophyte devel-opment. The juvenile gametophyte stage is initiated by the germina-

    tion of a haploid spore (circle) to form a chloronema filament (a).Under certain conditions, a tip chloronema cell will differentiate into

    a caulonema cell (marked by an arrowhead, b). c In the presence ofcytokinin a juvenile gametophyte undergoes transition indicated by

    the formation of a bud (marked by an arrowhead). This bud laterdevelops into an adult gametophyte or gametophore that bears

    gametangia (d). Upon egg fertilization a diploid sporophyte is formed(e, marked by an arrowhead). Relatively abundant miRNAs, whichare differentially expressed between the stages a, c and e (Table 1)are indicated next to the stage with the highest expression (Axtell

    et al. 2007)

    Plant Mol Biol


  • and Bowman (2004). They cloned the class III HD-ZIP

    gene homolog PpC3HDZIP1 from P. patens, and found

    that it contains a conserved miR166-target sequence.

    Using 50 rapid amplification of complementary DNA ends(50-RACE) they were able to clone a putative miR166-guided cleavage product of PpC3HDZIP1 mRNA (Floyd

    and Bowman 2004). This suggested that miR166 nega-

    tively regulates PpC3HDZIP1 expression in P. patens and

    provided indirect evidence for the presence of miR166 in

    this basal land plant.

    Soon after, the cloning of miR160 and miR160-guided

    cleavage products of auxin response factor (ARF) genes

    from the leafy gametophyte of the moss Polytrichum

    juniperinum was reported (Axtell and Bartel 2005). This

    provided direct evidence that miR160 is present and

    functional in moss. In A. thaliana, miR160 regulates

    ARF10, ARF16 and ARF17 (Liu et al. 2007; Mallory et al.

    2005; Wang et al. 2005) indicating that some miRNA:

    target interactions were deeply conserved during land plant


    Three groups used a conventional sRNA cloning

    approach to identify miRNAs from P. patens (Arazi et al.

    2005; Fattash et al. 2007; Talmor-Neiman et al. 2006a).

    Arazi et al. (2005) identified, among 100 cloned protonema

    (Fig. 1ac) sRNAs, homologs of miR390, miR156,

    miR319 and miR535, revealing their ancient origins within

    the land plant lineage. In addition, that study reported the

    cloning of five P. patens-specific miRNAs based on


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